Eukaryotic mRNA translation initiation is a complicated process involving assembly of a large protein-RNA complex that directs the ribosome to the initiation codon. Like transcription initiation, translation initiation represents a critical, rate-limiting step at which eukaryotic gene expression is regulated in response to developmental/environmental signals [reviewed in Mathews et al., "In Translational Control, eds. J. W. B. Hershey, M. B. Mathews, and N. Sonenberg. 1-29. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, (1996)]. For example, entry into and transit through the G1 phase of the cell cycle are correlated with increased rates of translation initiation [reviewed in Sonenberg et al., Proc. Natl. Acad. Sci. USA, 75:4843-4847 (1996)]. Eukaryotic mRNAs (excluding organellar mRNAs) are distinguished by the presence of a 5' cap structure and a 3' polyA tail that synergize in stimulating translation [reviewed in Shatkin, Cell, 9:645-653 (1976); Sachs and Wahle, J. Biol. Chem., 268:22955-22958 (1993)]. The cap consists of guanosine, methylated at position 7, connected by a 5' to 5' triphosphate bridge to the first nucleotide of the mRNA [7-methyl-G(5')ppp(5')N, where N is any nucleotide].
In the most general case (cap-dependent translation), protein synthesis begins with recognition of 7-methyl-G(5')ppp(5')N by eukaryotic initiation factor 4E (eIF4E or cap-binding protein). eIF4E is the least abundant of the general translation initiation factors, and is considered to be the factor limiting recruitment of the ribosome to the translation start-site [reviewed in Sonenberg, In Translational Control, eds. J. W. B. Hershey, M. B. Mathews, and N. Sonenberg, 245-269, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1996)]. Not surprising, overexpression of wild-type eIF4E in cultured cells causes malignant transformation [Lazaris-Karatzas et al., Nature, 345:544-547 (1990)]. eIF4E is a component of the eIF4F complex, which includes eIF4G (or p220) and eIF4A (an ATP-dependent RNA helicase). Biochemical studies revealed that eIF4G is a bridge between eIF4E and eIF4A [reviewed in Sonenberg, 1996, supra]. Following cap recognition by its eIF4E subunit, eIF4F and eIF4B unwind secondary structure in the 5'-untranslated region of the mRNA, rendering the initiation codon accessible to the ribosome [reviewed in Merrick and Hershey, In Translational Control, eds. J. W. B. Hershey, M. B. Mathews, and N. Sonenberg, 31-69, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1996)]. Thus, cap-binding by eIF4E establishes a stable protein-mRNA foundation for assembly of a functional translation initiation complex.
Given eIF4E's pivotal role in translation, it is not surprising that it is a critically-important target for regulation of gene expression in eukaryotes. The transcriptional activator c-myc regulates eIF4E levels via interactions with an E-box in the eIF4E gene promoter [Jones et al., Molec. Cell. Biol., 16:4754-4764 (1996)]. eIF4E activity is also regulated by post-translational modification, and by binding to negative regulators of translation initiation. In response to treatment of cells with growth factors, hormones and mitogens, mammalian eIF4E is phosphorylated at Ser209 [Joshi et al., J. Biol. Chem., 270:14597-14603 (1995); Whalen et al., J. Biol. Chem., 271:11831-11837 (1996)]. Phosphorylation increases eIF4E affinity for mRNA caps, thereby stimulating translation initiation in vivo [reviewed in Sonenberg, 1996, supra]. Conversely, eIF4E activity is suppressed by 4E-binding proteins, such as mammalian 4E-BP1, 4E-BP2 and 4E-BP3 [reviewed in Sonenberg, 1996, supra], and yeast p20 [Altmann et al., EMBO J., 16:1114-1121 (1997)]. These negative regulators of gene expression have no effect on cap-binding, but instead block interactions between eIF4E and eIF4G [Mader et al., Molec. Cell. Biol., 15:4990-4997 (1995); Haghighat et al., EMBO J., 14:5701-5709 (1995); Altmann et al., 1997, supra]. Therefore, the 4E-binding proteins repress cap-dependent translation by inhibiting assembly of the eIF4F complex (eIF4E, eIF4G, and eIF4A). Insulin (as well as other hormones, mitogens and growth factors) increases protein synthesis, at least in part, by relieving the repressive effect of 4E-BP1 [Lin et al., Science, 266:653-656 (1994); Pause et al., Nature, 371:762-767 (1994)], via the phosphatidylinositol 3-kinase signal transduction pathway [Manteuffel et al., Proc. Natl. Acad. Sci. USA, 93:4076-4080 (1996)]. When 4E-BP1 is phosphorylated it no longer forms a stable complex with eIF4E, and binding of eIF4G and assembly of a functional translation initiation complex can resume [reviewed in Sonenberg, 1996, supra].
eIF4E has been the focus of considerable biochemical and genetic study. After its identification [Sonenberg et al., 1978, supra] and initial purification [Sonenberg et al., Proc. Natl. Acad. Sci. USA, 76:4345-4349 (1979)], cDNAs encoding eIF4E were cloned from various eukaryotes. Sequence comparisons revealed a phylogenetically-conserved 182 amino acid C-terminal portion (FIG. 1). In contrast, the N-terminus varies in length, shows little or no conservation among different organisms and is not required for cap-dependent translation in vitro (see below). Current structural knowledge of eIF4E is limited to results from site-directed mutagenesis (see below), and a photoaffinity labeling study [Friedland et al., Protein Science, 6:125-131 (1996)]. The immediate challenge facing structural biologists interested in understanding translational regulation of gene expression is to establish the mechanistic and structural basis for eIF4E's interactions with the mRNA 5' cap, translation initiation factors, and regulatory proteins. This information is invaluable for the identification of methods of effecting these important translation initiation interactions, since translation initiation is a critical rate-limiting step in the regulation of eukaryotic gene expression response to developmental/environmental signals.
One such means of effecting the eIF4E protein and thereby, eukaryotic gene expression in general, is to identify agonists or antagonists to the eIF4E protein. Unfortunately, such identification has heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. A far superior method of drug-screening relies on structure based drug design. In this case, the three dimensional structure of a protein-inhibitor complex is determined and potential agonists and/or potential antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, Dec.:92-98 (1993); West et al., TIPS, 16:67-74 (1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)]. However, heretofore the three-dimensional structure of the eIF4E protein has remained unknown, essentially because no eIF4E protein crystals had been produced of sufficient quality to allow the required X-ray crystallographic data to be obtained. Therefore, there is presently a need for obtaining a form of the eIF4E protein that can be crystallized with a ligand (such as an inhibitor) to form a crystal with sufficient quality to allow such crystallographic data to be obtained. Further, there is a need for such crystals. Furthermore there is a need for the determination of the three-dimensional structure of such crystals. Finally, there is a need for procedures for related structural based drug design based on such crystallographic data.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.